Determination of emodin in Polygonum cuspidatum by HPLC
Time: 2022-02-17 11:52:27
Abstract: Polygonum cuspidatum is not only for viewing, but also food. The tender stems are used as vegetables, and the roots are used as cold drinks. It is placed in cold water to cool down. Rice noodles have a unique flavor. It is also called "sour soup stick" because of its sour taste.
Polygonum cuspidatum was included in the first edition of the 2000 edition of the Chinese Pharmacopoeia, and the control content of total quinone was determined by spectrophotometry. Polygonum cuspidatum contains anthraquinone derivatives, mainly in free form, including emodin, emodin methyl ether, chrysophanol and other components. In this paper, high performance liquid chromatography was used to determine the content of emodin in the medicinal materials of Polygonum cuspidatum. The method is fast, convenient and convenient. reliable.
1 Instruments and reagents
Agilent 1100 combined automatic high performance liquid chromatograph. Polygonum cuspidatum (commercially available), emodin reference substance (China Institute for the Control of Pharmaceutical and Biological Products, for content determination), methanol is of chromatographic grade, and other reagents are of analytical grade.
2 Methods and results
2.1 Chromatographic conditions
Chromatographic column: KromasilC18 (250mm×4.6mm, 5μm), mobile phase: methanol-0.1% phosphoric acid solution (85:15), flow rate: 1.0mlmin, column temperature: room temperature, measurement wavelength: 254nm, detected by external standard method, input Sample volume: 5.0 μL.
2.2 Preparation of the test solution
Take about 0.5g of this product powder (passed through a No. 3 sieve) (at the same time, take another powder of this product to measure the loss on drying at 105°C), accurately weigh it, put it in a 100ml volumetric flask, add about 80ml of methanol, ultrasonically treat it (power 250W, frequency 30kHz) 30min, let cool to room temperature, add methanol to the mark, shake well, filter, accurately measure 10ml of the subsequent filtrate, put it in a 100ml round-bottom flask, evaporate methanol, add 20ml of 2.5mol/L sulfuric acid solution, and heat under reflux for 1h , cooled, added 30nll of chloroform, continued to reflux for 2h, separated the chloroform layer, extracted the acid solution with chloroform 3 times, 5ml each time, combined the chloroform extracts, passed through a funnel equipped with anhydrous sodium sulfate, placed in a 50ml volumetric flask, the filter Wash with a small amount of chloroform, add the washing solution to the filtrate, add chloroform to the mark, shake well, accurately measure 10ml of chloroform solution, evaporate to dryness in a water bath, add an appropriate amount of methanol to the residue to dissolve, put it in a 25ml volumetric flask, add methanol to dilute to the mark, Shake well, as the test solution.
2.3 Preparation of reference solution
Accurately weigh 9.81mg of emodin reference substance, put it in a 100ml volumetric flask, add an appropriate amount of methanol to dissolve, and add methanol to the mark, shake well, and use it as a reference substance stock solution (concentration is 0.0981mg/ml). Precisely measure 1ml, add methanol to dilute to 25ml, as the reference solution.
2.4 Investigation of Linear Relationship
The above emodin reference solution was injected into 2.0 μL, 4.0 μL, 6.0 μL, 8.0 μL, 10.0 μL and 12.0 μL respectively. Record the peak area, plot the peak area (Y) against the injection amount (X), get the standard curve, and calculate the regression equation: Y=4.0387×103X−0.844, r=0.9999, and the linear range is 7.848×10-3～4.709 ×10-2.
2.5 Precision test
Take the same test solution, repeat the injection 6 times, record the peak area, the RSD of the result is 0.2%, indicating that the precision is good.
2.6 Repeatability test
Take 5 samples of the same batch of Polygonum cuspidatum, measure the content of emodin according to the above method, and the RSD is 0.5%.
2.7 Solution stability test
Take the test solution under item "2.6", inject 5μL at 0, 4, 8, 12, 16, 20, and 24h respectively, and record the peak area. The RSD of the peak area is 0.9%, indicating that the test solution is in 24h. Internally stable.
2.8 Recovery test
Accurately weigh an appropriate amount of the sample with a known content of 1.6%, add 15ml of the stock solution of emodin reference substance precisely, measure it according to the above method, and calculate the sample recovery rate.
2.9 Sample determination
The content of emodin in the samples was determined by taking the medicinal materials of Polygonum cuspidatum according to the above method, and the results are shown in Table 2. The reference substance solution and the test substance solution were injected into sva respectively. According to this method, the sample and the reference substance had a corresponding chromatographic peak at the same retention time position.
Take the emodin reference solution and scan it in the wavelength range of 200-400nm. The results show that there are maximum absorption peaks at 219nm, 254nm and 292nm. Since the absorption at 254nm is strong and not disturbed by the solvent peak, 254nm is selected. As the measured absorption wavelength, it is consistent with the provisions of "Chinese Pharmacopoeia".
Experiments show that after this product is hydrolyzed and then extracted with chloroform for 4 times, the emodin has been basically completely extracted.
It can be seen from the test results of linearity, recovery rate, reproducibility and solution stability that the HPLC method is used to determine the content of emodin in Polygonum cuspidatum, with high accuracy, good reproducibility, and simple and fast operation.